The only residue in eukaryotic DNA which is modified after polymerization is cytosine which is methylated to give 5-methylcytosine (m5C). Although the average mammalian cell has approximately 4% of its cytosine residues methylated and significant changes in the amount of DNA cytosine methylation have been reported from several different types of cancer cells, extremely little is known about the influence of methylation of DNA cytosine on the physiology of normal or cancerous cells. We have recently developed two high pressure liquid chromatography HPLC methods for terming the mol % m5C in unlabeled DNA as well as its base composition; these two methods are the most sensitive reported to date. One of these will be used to quantitate m5C levels in DNA from various normal tissues and cell cultures of human, murine, or bovine origin. Also, DNA from many different types of tumor tissue and two types of differentiating cells will be examined. The intragenomic distribution of m5C will be studied by determining the mol% m5C (and base composition) of DNA fractionated according to kinetic reassociation complexity, buoyant density in cesium salt gradients, DNase I-digest dinucleotide composition, pyrimidine isoplith composition, and mitochondrial vs. nuclear origin and resistance to specific restriction endonucleases. We will also try to isolate from mammalian cells DNA-binding proteins which specifically bind to m5C-rich DNA or regions in DNA. We will use bacteriophage XP-12 DNA as a uniquely m5C-rich DNA to isolate and assay such proteins. It is postulated that such proteins have critical roles to play in controlling transcription or DNA replication and, thereby, in certain phases of differentiation and oncogenic transformation.